Keisuke Shibata

Microglia contact induces synapse formation in developing somatosensory cortex

Microglia are the immune cells of the central nervous system that play important roles in brain pathologies. Microglia also help shape neuronal circuits during development, via phagocytosing weak synapses and regulating neurogenesis. Using in vivo multiphoton imaging of layer 2/3 pyramidal neurons in the developing somatosensory cortex, we demonstrate here that microglial contact with dendrites directly induces filopodia formation. This filopodia formation occurs only around postnatal day 8–10, a period of intense synaptogenesis and when microglia have an activated phenotype. Filopodia formation is preceded by contact-induced Ca2+ transients and actin accumulation. Inhibition of microglia by genetic ablation decreases subsequent spine density, functional excitatory synapses and reduces the relative connectivity from layer 4 neurons. Our data provide the direct demonstration of microglial-induced spine formation and provide further insights into immune system regulation of neuronal circuit development, with potential implications for developmental disorders of immune and brain dysfunction.

Cortical astrocytes rewire somatosensory cortical circuits for peripheral neuropathic pain

Long-term treatments to ameliorate peripheral neuropathic pain that includes mechanical allodynia are limited. While glial activation and altered nociceptive transmission within the spinal cord are associated with the pathogenesis of mechanical allodynia, changes in cortical circuits also accompany peripheral nerve injury and may represent additional therapeutic targets. Dendritic spine plasticity in the S1 cortex appears within days following nerve injury; however, the underlying cellular mechanisms of this plasticity and whether it has a causal relationship to allodynia remain unsolved. Furthermore, it is not known whether glial activation occurs within the S1 cortex following injury or whether it contributes to this S1 synaptic plasticity. Using in vivo 2-photon imaging with genetic and pharmacological manipulations of murine models, we have shown that sciatic nerve ligation induces a re-emergence of immature metabotropic glutamate receptor 5 (mGluR5) signaling in S1 astroglia, which elicits spontaneous somatic Ca2+ transients, synaptogenic thrombospondin 1 (TSP-1) release, and synapse formation. This S1 astrocyte reactivation was evident only during the first week after injury and correlated with the temporal changes in S1 extracellular glutamate levels and dendritic spine turnover. Blocking the astrocytic mGluR5-signaling pathway suppressed mechanical allodynia, while activating this pathway in the absence of any peripheral injury induced long-lasting (>1 month) allodynia. We conclude that reawakened astrocytes are a key trigger for S1 circuit rewiring and that this contributes to neuropathic mechanical allodynia.

Grape Seed Extract Acting on Astrocytes Reveals Neuronal Protection Against Oxidative Stress via Interleukin-6-mediated Mechanisms

Grape polyphenols are known to protect neurons against oxidative stress. We used grape seed extract (GSE) from “Koshu” grapes (Vitis vinifera) containing a variety of polyphenols, and performed transcriptome analysis to determine the effects of GSE on primary cultures of astrocytes in the hippocampus. GSE upregulated various mRNAs for cytokines, among which interleukin-6 (IL-6) showed the biggest increase after treatment with GSE. The GSE-evoked increase in IL-6 mRNAs was confirmed by quantitative RT-PCR. We also detected IL-6 proteins by ELISA in the supernatant of GSE-treated astrocytes. We made an oxidative stress-induced neuronal cell death model in vitro using a neuron rich culture of the hippocampus. Treatment of the neurons with H2O2 caused neuronal cell death in a time- and concentration-dependent manner. Exogenously applied IL-6 protected against the H2O2-induced neuronal cell death, which was mimicked by endogenous IL-6 produced by GSE-treated astrocytes. Taken together, GSE acting on astrocytes increased IL-6 production, which functions as a neuroprotective paracrine, could protect neuronal cells from death by oxidative stress.

Urothelial ATP exocytosis: Regulation of bladder compliance in the urine storage phase

The bladder urothelium is more than just a barrier. When the bladder is distended, the urothelium functions as a sensor to initiate the voiding reflex, during which it releases ATP via multiple mechanisms. However, the mechanisms underlying this ATP release in response to the various stretch stimuli caused by bladder filling remain largely unknown. Therefore, the aim of this study was to elucidate these mechanisms. By comparing vesicular nucleotide transporter (VNUT)-deficient and wild-type male mice, we showed that ATP has a crucial role in urine storage through exocytosis via a VNUT-dependent mechanism. VNUT was abundantly expressed in the bladder urothelium, and when the urothelium was weakly stimulated (i.e. in the early filling stages), it released ATP by exocytosis. VNUT-deficient mice showed reduced bladder compliance from the early storage phase and displayed frequent urination in inappropriate places without a change in voiding function. We conclude that urothelial, VNUT-dependent ATP exocytosis is involved in urine storage mechanisms that promote the relaxation of the bladder during the early stages of filling.

Clock Genes Regulate the Circadian Expression of Piezo1, TRPV4, Connexin26, and VNUT in an Ex Vivo Mouse Bladder Mucosa

Objectives ClockΔ19/Δ19 mice is an experimental model mouse for nocturia (NOC). Using the bladder mucosa obtained from ClockΔ19/Δ19 mice, we investigated the gene expression rhythms of mechanosensory cation channels such as transient receptor potential cation channel subfamily V member 4 (TRPV4) and Piezo1, and main ATP release pathways including vesicular nucleotide transporter (VNUT) and Connexin26(Cx26), in addition to clock genes. Materials and methods Eight- to twelve-week-old male C57BL/6 mice (WT) and age- and sex-matched C57BL/6 ClockΔ19/Δ19 mice, which were bred under 12-h light/dark conditions for 2 weeks, were used. Gene expression rhythms and transcriptional regulation mechanisms in clock genes, mechanosensor, Cx26 and VNUT were measured in the mouse bladder mucosa, collected every 4 hours from WT and ClockΔ19/Δ19 mice using quantitative RT-PCR, a Western blot analysis, and ChIP assays. Results WT mice showed circadian rhythms in clock genes as well as mechanosensor, Cx26 and VNUT. Their expression was low during the sleep phase. The results of ChIP assays showed Clock protein binding to the promotor regions and the transcriptional regulation of mechanosensor, Cx26 and VNUT. In contrast, all of these circadian expressions were disrupted in ClockΔ19/Δ19 mice. The gene expression of mechanosensor, Cx26 and VNUT was maintained at a higher level in spite of the sleep phase. Conclusions Mechanosensor, Cx26 and VNUT expressed with circadian rhythm in the mouse bladder mucosa. The disruption of circadian rhythms in these genes, induced by the abnormalities in clock genes, may be factors contributing to NOC because of hypersensitivity to bladder wall extension.

The Clock mutant mouse is a novel experimental model for nocturia and nocturnal polyuria.

The pathophysiologies of nocturia (NOC) and nocturnal polyuria (NP) are multifactorial and their etiologies remain unclear in a large number of patients. Clock genes exist in most cells and organs, and the products of Clock regulate circadian rhythms as representative clock genes. Clock genes regulate lower urinary tract function, and a newly suggested concept is that abnormalities in clock genes cause lower urinary tract symptoms. In the present study, we investigated the voiding behavior of Clock mutant (ClockΔ19/Δ19) mice in order to determine the effects of clock genes on NOC/NP.

An effective therapeutic approach for oxaliplatin-induced peripheral neuropathy using a combination therapy with goshajinkigan and bushi

ABSTRACT Oxaliplatin-induced peripheral neuropathy (OIPN) occurs at extraordinarily high frequency, but no effective treatment for this disorder has been established. Goshajinkigan (GJG), a traditional Japanese medicine known as Kampo, is known to reduce OIPN in both basic and clinical studies. However, its molecular mechanisms remain largely unknown. Here, we elucidate the mechanisms underlying the therapeutic effects of GJG against OIPN and the therapeutic benefits of combining GJG with bushi, a herbal medicine derived from the processed Aconiti tuber. Oxaliplatin (4 mg/kg) was injected into mice twice a week for up to 4 and 3 weeks, respectively. OIPN was assessed using pain behavioral tests, such as those testing cold hypersensitivity, thermal hyperalgesia, and mechanical allodynia, as well as a reduction of the current perception threshold (CPT). GJG (0.3 or 1 g/kg) and bushi (0.1 or 0.3 g/kg) were orally administered 5 times a week for 4 weeks. Behavioral analysis was performed 24 h after the final dose. Oxaliplatin induced cold hypersensitivity and mechanical allodynia but not thermal hyperalgesia and reduced CPT of Aδ- and Aβ-fibers but not C-fibers. All these effects were counteracted by GJG. Bushi, an ingredient of GJG that shows analgesic effect, reduced oxaliplatin-induced cold hypersensitivity but had no effect on oxaliplatin-induced mechanical allodynia. However, bushi significantly accentuated the effects of GJG when co-administered with GJG. GJG reduces OIPN by counteracting the sensitization of Aδ- and Aβ-fibers and shows analgesic effects against cold hypersensitivity and mechanical allodynia. These effects are potentiated by bushi. The combination of GJG with bushi has high potential for preventing OIPN.

The Circadian expression of Piezo1, TRPV4, Connexin26, and VNUT, associated with the expression levels of the clock genes in mouse primary cultured urothelial cells

To investigate circadian gene expressions in the mouse bladder urothelium to establish an experimental model and study the functions of the circadian rhythm.

P2Y 6 -deficiency increases micturition frequency and attenuates sustained contractility of the urinary bladder in mice

The role of the P2Y6 receptor in bladder function has recently attracted a great deal of attention in lower urinary tract research. We conducted this study to determine contributions of the P2Y6 receptor in lower urinary tract function of normal phenotypes by comparing P2Y6-deficient mice and wild-type mice. In in vivo experiments, P2Y6-deficient mice had more frequent micturition with smaller bladder capacity compared to wild-type mice; however, there was no difference between these groups in bladder-filling pressure/volume relationships during cystometry under decerebrate, unanaesthetized conditions. Analysis of in vivo bladder contraction revealed significant difference between the 2 groups, with P2Y6-deficient mice presenting markedly shorter bladder contraction duration but no difference in peak contraction pressure. However, analysis of in vitro experiments showed no P2Y6 involvements in contraction and relaxation of bladder muscle strips and in ATP release by mechanical stimulation of primary-cultured urothelial cells. These results suggest that the P2Y6 receptor in the central nervous system, dorsal root ganglion, or both is involved in inhibition of bladder afferent signalling or sensitivity in the pontine micturition centre and that the receptor in the detrusor may be implicated in facilitation to sustain bladder contraction force.

Anti-Depressant Fluoxetine Reveals its Therapeutic Effect Via Astrocytes

Although psychotropic drugs act on neurons and glial cells, how glia respond, and whether glial responses are involved in therapeutic effects are poorly understood. Here, we show that fluoxetine (FLX), an anti-depressant, mediates its anti-depressive effect by increasing the gliotransmission of ATP. FLX increased ATP exocytosis via vesicular nucleotide transporter (VNUT). FLX-induced anti-depressive behavior was decreased in astrocyte-selective VNUT-knockout mice or when VNUT was deleted in mice, but it was increased when astrocyte-selective VNUT was overexpressed in mice. This suggests that VNUT-dependent astrocytic ATP exocytosis has a critical role in the therapeutic effect of FLX. Released ATP and its metabolite adenosine act on P2Y11 and adenosine A2b receptors expressed by astrocytes, causing an increase in brain-derived neurotrophic factor in astrocytes. These findings suggest that in addition to neurons, FLX acts on astrocytes and mediates its therapeutic effects by increasing ATP gliotransmission.